ORGANIC LETTERS
Water-Induced Switching of β-Structure to Polyproline II Conformation in the 4S-Aminoproline Polypeptide via H-Bond Rearrangement
2010 Vol. 12, No. 23 5390-5393
Mahesh V. Sonar† and Krishna N. Ganesh*,†,‡ DiVision of Organic Chemistry, National Chemical Laboratory, Dr Homi Bhabha Road, Pune 411008, India, and Indian Institute of Science Education and Research, 900, NCL InnoVation Park, Dr Homi Bhabha Road, Pune 411008, India
[email protected] Received September 14, 2010
ABSTRACT
4S-Aminoproline polypeptide 2 forms unusual β-structure in trifluoroethanol that switches to the polyproline II (PPII) form in aqueous medium, while 4R-aminoproline peptide 1 retains PPII form in both solvents. This first instance of a polyproline derivative showing a β-structure is attributed to competitive pH-dependent (4-NH3+/NH2) stereoelectronic effect (4R vs 4S) and the overriding importance of stereospecific intra/ intermolecular H-bonding in (2,4)-cis-4S-aminoproline in contrast to (2,4)-trans-4R-aminoproline oligomers.
The polyproline type II (PPII) helix is a prevalent conformation in both folded and unfolded proteins1 and plays an important role in a wide variety of biological processes, such as signal transduction, transcription, immune response, and cell motility.2 Each strand of collagen triplex with the ProHyp-Gly tripeptide repeat unit adopts a left-handed PPIIlike conformation.3 Oligoprolines and their derivatives have
found utility as cell penetrating agents4 and as molecular spacers in biomimetic systems for energy/electron transport.5 The PPII helix is a fully extended left-handed structure with all amide bonds in the trans conformation, while the righthanded PPI helix is compact, with all amide bonds in the cis conformation.1 It is well-known that polyprolines adopt PPII conformation in water6 and PPI conformation in hydrophobic solvents of short chain aliphatic alcohols.6 It has been well demonstrated that the stereoelectronic effect
†
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10.1021/ol1021993 2010 American Chemical Society Published on Web 11/04/2010
(4) (a) Farrera-Sinfreu, J.; Giralt, E.; Castel, S.; Albericio, F.; Royo, M. J. Am. Chem. Soc. 2005, 127, 9459–9468. (b) Fillon, Y. A.; Anderson, J. P.; Chmielewski, J. J. Am. Chem. Soc. 2005, 127, 11798–11803. (5) (a) Doose, S.; Neuweiler, H.; Barsch, H.; Saer, M. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 17400–17405. (b) Schuler, B.; Lipman, E. A.; Steinbach, P. J.; Klumke, M.; Eaton, W. A. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 2754–2759. (6) (a) Knof, S.; Engel, J. Isr. J. Chem 1974, 12, 165–177. (b) Mutter, M.; Wohr, T.; Gioria, S.; Keller, M. Biopolymers 1999, 51, 121–128. (c) Kakinoki, S.; Hirano, Y.; Oka, M. Polym. Bull. 2005, 53, 109–115.
of the 4-substituent plays a major role in determining the pucker of the pyrrolidine ring of proline and hence the conformational stability of proteins.7 In this context, we reported earlier that 4-amino substitution on proline in collagen peptide stabilizes the triple helix8 at both acidic and basic pHs. In collagen, the tripeptide repeat unit [Pro-Hyp-Gly] has glycine whose amide linkage is involved in an interchain H-bond, leading to the triple helix structure. In contrast, polyproline peptides lack amide NH and hence are unable to form a triplex via interchain H-bonds, ending up as a single helix of PPI or PPII type. Recently, 4S(OH/NH3+) groups on proline were shown to form intramolecular H-bonds with the amide carbonyl, increasing the trans/cis amide ratio and thereby promoting PPII conformation in the derived polypeptides.9 Unlike other 4-substituents on proline studied so far (OH, SH, CH3, F), the ionizable 4-NH2 group is a good probe to examine the pH effects on polyproline conformation. Herein, we report the novel behavior of 4S-aminoproline polypeptide to form a novel β-structure in trifluoroethanol (TFE) that switches to PPII form in aqueous medium. This property exclusive to 4Saminoproline polypeptide arises from a stereospecific intramolecular H-bonding that stabilizes the PPII form, while the unusual β-structure results from interchain H-bonding. To our knowledge, this is perhaps the first report of formation of β-structure in any polyproline derivatives and its switch over to PPII form induced by water. Synthesis and Conformational Studies of 4-Aminoproline Oligomers. The oligopeptides 1-3 were synthesized from appropriate N-Fmoc-protected monomers asssembled in the solid phase, purified by HPLC, and characterized by MALDI-TOF (for details, see Supporting Information). The CD spectral analyses were carried out as a function of temperature, pH, urea, and solvents (buffer and trifluoroethanol, TFE). All three peptides (100 µM, pH 7.2) show CD spectra (Figure 1) with a positive band between 220 and 230 nm and a negative band between 200 and 210 nm that are the established patterns of the PPII conformation.10 The intensity of the positive band at 225-227 nm is proportional to the PPII helical content which is seen to decrease in the order 4R-Amp9 1 > 4S-amp9 2 > Pro9 3. The effect of protonation of the 4-amino group on PPII helical content was examined by the CD spectra of peptides 1-3 (Figure 2A) recorded at different pH (4.0-10.0). The positive ellipticity at 225 nm for 4R-Amp9 1 decreased by 10% with increasing pH up to 7.2 and did not change further until pH 10.0. In the case of 4S-amp9 2, positive intensity was enhanced in the pH range 4.0-10.0 in a sigmoidal (7) Bretscher, L. E.; Jenkins, C. L.; Taylor, K. L.; DeRider, M. L.; Raines, R. T. J. Am. Chem. Soc. 2001, 123, 777–778. (8) Babu, I. R.; Ganesh, K. N. J. Am. Chem. Soc. 2001, 1232079-2080. (b) Umashankara, M.; Babu, I. R.; Ganesh, K. N. Chem. Commun. 2003, 2606–2607. (9) (a) Shoulders, M. D.; Kotch, F. K.; Choudhary, A.; Guzei, I. A.; Raines, R. T. J. Am. Chem. Soc. 2010, 132, 10857–10865. (b) Kuemin, M.; Nagel, Y. A.; Schweizer, S.; Monnard, F. W.; Ochsenfeld, C.; Wennemers, H. Angew. Chem., Int. Ed. 2010, 49, 6324–6327. (10) Woody, R. W. AdV. Biophys. Chem. 1992, 2, 37–79. Org. Lett., Vol. 12, No. 23, 2010
Figure 1. CD profiles of polypeptides 1, 4R-Amp (b); 2, 4S-amp (2); and 3, Pro9 (9), all at 100 µM (pH 7.2).
fashion. At acidic pH (4.0-5.0), the PPII helicity of 4Samp9 2 was low (20% of 4R-Amp9 1) but increased by 2-fold at pH 10.0. The ellipticity of peptide Pro9 3 remained
Figure 2. (A) Intensity of the positive band of CD spectra of peptides 1-3 as a function of pH. (B) Thermal stability of PPII helices in peptides 1-3 as a function of pH, followed at 225 nm.
constant with pH. This suggests a pivotal role for both stereochemistry and the protonation status of the 4-amino group in eliciting the PPII helicity of 4(R/S)-aminoproline polypeptides 1 and 2. The pH-dependent thermal stability (Tm) of PPII helices in peptides 1-3 was measured from temperature-dependent CD spectral data (Figure 2B and Supporting Information). It is seen that (i) 4R-Amp9 1 has maximum Tm at all pHs; almost invariant (ii) 4S-amp9 2 has the lowest Tm among the peptides at pH 4.0 but increased gradually with raise in pH to 10.0 to a value closer to the Tm of 4R-Amp9 1; and (iii) Pro9 3 with intermediate Tm at pH 4.0 remained constant over the pH range. The 4-NH3+ group at pH 4.0 stabilized the PPII helix most in the 4R-form and least in the 4S-form, while 4-NH2 at pH 10.0 stabilized both 4R- and 4S-peptides to a similar extent. The 4S-amp9 2 thus exhibited significant pH-dependent PPII stability that is maximum in the unionized amino form. 5391
Figure 3. (A) CD spectra of peptides 1-3 in trifluoroethanol (TFE). (B) CD spectra of 4S-amp9 2 in TFE with incremental addition of phosphate buffer (pH 7.2) from (a) 0.1% to (k) 1.0% in 0.1% steps and (l) 2.0%. (C) Increasing concentration of 2 from 50 to 250 µM.
Urea is known to enhance the PPII helical content through rigidification of the polypeptide backbone.11 The low PPII helicity of 4S-amp9 2 was enhanced enormously (>300%) by addition of 1 M urea (pH 7.2), while that of 4R-Amp9 1 and (Pro9) 3 increased by a mere 15-20% (Supporting Information). The larger changes seen specifically for 4Samp9 2 in the presence of urea and upon increasing the pH suggest the combined role of H-bonding and stereoelectronic12 effects in dictating the PPII conformation. Solvent plays a key role in modulating the H-bonding effects, and hence the CD spectra of peptides 1-3 were recorded in a flourinated solvent trifluoroethanol (TFE) (Figure 3A). The 4R-Amp9 1 and Pro9 3 show CD spectra typical of PPII form. Very interestingly, the CD spectra of 4S-amp9 2 in TFE are unlike the PPII pattern, showing a negative maximum around 214 nm and a broad shoulder at 228 nm that is typical of β-structure.13 When aqueous phosphate buffer (pH 7.2) was titrated into TFE solution of 4S-peptide 2 in tiny incremental steps of 0.1%, the 214 nm negative band slowly shifted to 205 nm, accompanied by a growing of the broad negative shoulder at 228 nm into a positive band at about 224 nm (Figure 3B) typical of PPII form. The isosbestic point seen at 215 nm is indicative of the conversion of 4S-peptide 2 from β-structure in 100% TFE to full PPII form with 0.8% buffer in TFE. Upon increasing the concentration of 4S-amp9 2 from 50 to 250 µM in TFE, the CD spectra exhibited a nice growth in the negative band intensity at 210 nm, accompanied by its shift to 216 nm and a large increase of the positive band at 200 nm (Figure 3C). As expected for intermolecular hydrogen bonding increasing with concentration, this strongly points to a consolidation of β-structure in the peptide 4Samp9 2. In the case of 4R-Amp9 1, increasing the peptide concentration leads to enhancement of the PPII form without any other changes. The overall results imply that 4S-amp9 2 assumes a β-structure in TFE that is transformed to the PPII form in aqueous medium, unlike 4R-amp9 1 which retains the PPII form in both conditions. (11) (a) Robinson, D. R.; Jencks, W. P. J. Am. Chem. Soc. 1965, 87, 2462–2469. (b) Whittington, S. J.; Chellgren, B. W.; Hermann, V. M.; Creamer, T. P. Biochemistry 2005, 44, 6269–6275. (12) Jia-Cherng, H.; Raines, R T. Protein Sci. 2006, 15, 74–83. (13) Seebach, D.; Overhand, M.; Kiihnle, F. N. M.; Martinoni, B. HelV. Chim. Acta 1996, 79, 913–941. 5392
The formation of β-structure in polyproline peptides under any conditions is unprecedented in the literature since they lack H-bond donor sites. In 4S-amp9 2, the NH2 group can form an intramolecular H-bond with the amide carbonyl of the same proline moiety, promoting a PPII conformation.9b The possibility of the 4S-NH2 group engaging the amide carbonyl of another chain of 4S-amp9 through an intermolecular H-bond would lead to β-structure. Such an interchain H-bonded structure should be facilitated at higher peptide concentration. A plausible molecular picture for this conversion is depicted in Scheme 1. The intramolecular H-bonding of 4S-
Scheme 1. Solvent-Derived Rearrangement of H-Bonds in 4S-amp6 Leading to Conformational Switch
NH2 with amide carbonyl possible only in 4S-amp9 2 (I) promotes the PPII form in buffer. Urea rigidifies the backbone11 by complementary H-bonding (II) among the cisdisposed 4S-NH2 group and the amide carbonyl to strengthen the PPII form. In a fluorinated solvent TFE, the intramolecular H-bonding between 4S-NH2 and the amide carbonyl switches to interchain H-bonding (III) giving rise to antiparallel β-structure. The trans disposition of 4R-NH2 and Org. Lett., Vol. 12, No. 23, 2010
the amide carbonyl group in 4R-Amp9 (IV) is not conducive to formation of either intramolecular or strong interchain H-bonding in the derived peptide. Stereoelectronic vs H-Bonding Effects in 4S-amp9 2. The stereoelectronic effect of 4R-X substituents on proline is known to strongly favor the PPII form, over that of 4S-X substituents by enhancing the trans-amide content.12 Although the intramolecular H-bonding in 4S-NH3+ favors the trans-amide form,9b the unfavorable stereoelectronic effect of 4S-NH3+ (unlike 4R-NH3+) strongly negates the benefit of H-bonding (V), leading to a low PPII form for 2 at acidic pH. At higher pH, the effect of intramolecular H-bonding dominates a weaker stereoelectronic effect of 4S-NH2, promoting higher PPII content in peptide 2. While the conversion of PPII to PPI takes many hours/days due to a slow conversion of amide from trans to cis form,14 the switching of β-structure to PPII form is fast, within minutes, suggesting that the amide bond of 2 is also in trans form in the β-structure. No PPI form was seen for any of the peptides 1-3, under different conditions of pH, n-propanol and TFE. In conclusion, it is demonstrated here that 4S-amp9 2 adapts an unusual β-structure in TFE unlike most polyproline peptides which prefer the PPI form in hydrophobic/ fluorinated media. The β-structure arises from interchain (14) Chiang, Y.-C.; Lin, Y.-J.; Horng, J.-C. Protein Sci. 2009, 18, 1967– 1977.
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hydrogen bonds involving 4S-NH2 and amide carbonyl, which are broken in water and rearranged to intramolecular H-bonding that favors the PPII form via enriching the transamide geometry. This structural conversion illustrates a fine balance between stereoelectronic and H-bonding effects in novel tuning of the secondary structure of 4R/S-aminoproline polypeptides. β-Structure in polyproline peptides is hitherto unknown, and the present results will add a new design principle to a growing repertoire of strategies for engineering peptide secondary structural motifs for new biomaterials and nanoassemblies.15 Acknowledgment. MVS acknowledges an award of fellowship by Lady Tata Memorial Trust and CSIR (New Delhi). KNG holds JC Bose Fellowship of DST (New Delhi). Supporting Information Available: Experimental synthesis procedures, HPLC, mass spectral data, and CD spectra of peptides 1-3 under different conditions of pH, urea, salt, and concentration. This material is available free of charge via the Internet at http://pubs.acs.org. OL1021993 (15) (a) Tsai, C.-J.; Zheng, J.; Zanuy, D.; Haspel, N.; Wolfson, H.; Alema’n, C.; Nussinov, R. Proteins 2007, 68, 1–12. (b) Zhao, X.; Pan, F.; Xu, H.; Yaseen, M.; Shan, H.; Hauser, C. A. E.; Zhang, S.; Lu, J. R. Chem. Soc. ReV. 2010, 39, 3480–3498. (c) Apostolovic, B.; Maarten, D. M.; Klok, H.-M. Chem. Soc. ReV. 2010, 39, 3541–3550.
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